Much attention and effort has been applied in recent years to the production of fuels and chemicals, primarily ethanol, from cellulosic feedstocks, such as agricultural wastes and forestry wastes, due to their low cost and wide availability. These agricultural and forestry wastes are typically burned and landfilled; thus, using these cellulosic feedstocks for ethanol production offers an attractive alternative to disposal.
The first chemical processing step for converting cellulosic feedstock to ethanol or other fermentation products usually involves pretreatment of the feedstock. The purpose of the pretreatment is to increase the cellulose surface area, with limited conversion of the cellulose to glucose. Pretreatment of the feedstock can be achieved using an acid pretreatment conducted under conditions that hydrolyse the hemicellulose component of the feedstock, followed by enzymatic hydrolysis of the cellulose remaining in the pretreated cellulosic feedstock with cellulase enzymes. Enzymatic hydrolysis is typically conducted in one or more dilute mixed batch reactors under controlled pH, temperature and mixing conditions.
The cellulase enzymes utilized to hydrolyze the cellulose to glucose include a mix of enzymes including exo-cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidases. The CBH and EG enzymes catalyze the hydrolysis of the cellulose (β-1,4-D-glucan linkages). The CBH enzymes, CBHI and CBHII (also known as Cel7 and Cel6 according to Glycoside Hydrolase family designations), act on the ends of the glucose polymers in cellulose microfibrils and liberate cellobiose, while the EG enzymes (including EGI, EGII, EGIII and EGV, also known as Cel7, Cel5, Cel12 and Cel45, respectively) act at random locations on the cellulose. Together, the cellulase enzymes hydrolyze cellulose to cellobiose, which, in turn, is hydrolyzed to glucose by beta-glucosidase (beta-G).
In addition to CBH, EG and beta-glucosidase, there are several accessory enzymes that aid in the enzymatic digestion of cellulose (see co-owned WO 2009/026722 (Scott), which is incorporated herein by reference, and Harris et al., 2010, Biochemistry, 49:3305-3316). These include EGIV, also known as Cel61, swollenin, expansin, lucinen and cellulose-induced protein (Cip). Glucose can be enzymatically converted to the dimers gentiobiose, sophorose, laminaribiose and others by beta-glucosidase via transglycosylation reactions.
In conventional hydrolysis reactors, mixing is provided by mechanical mixers such as top-mounted, side-mounted, or bottom-mounted impellers, agitators or eductors; rapid movement of liquid slurry streams pumped into or through the vessel; and/or introducing or generating gases or vapours in the vessel. Moreover, reactors are known that employ periodic mixing as the slurry passes through mixing zones along the length of the reactor (see U.S. Pat. No. 5,733,758 (Nguyen) discussed below).
The fermentation to produce ethanol from the glucose is typically carried out with a Saccharomyces spp. strain. Recovery of the ethanol is achieved by distillation and the ethanol is further concentrated by molecular sieves.
The addition of water to the incoming feedstock to form a slurry facilitates the transportation and mechanical handling of the cellulosic feedstock. The slurry consists of cellulosic feedstock pieces or particles in water. Typically, the mass of water present is at least 5 to 25 times the mass of feedstock solids present for the slurry to flow uniformly.
However, the processing of slurries containing such high water content has certain drawbacks in plant operations. For example, during acid pretreatment, the high water content in the incoming slurry requires a large amount of steam for the heat-up, as well as acid. Moreover, large volumes of water in the slurry result in increases in equipment size, which, in turn, increases capital cost.
WO 2010/022511 (Anand et al.) discloses a process involving the removal of a significant amount of water from a cellulosic feedstock slurry by a pressurized screw press prior to its heat-up by steam in a pretreatment reactor. Advantageously, due to the high solids content attained by pressing, less liquid needs to be heated, thereby reducing the amount of steam required during the subsequent pretreatment. Moreover, a concentrated slurry can also reduce the amount of acid or alkali that is needed to catalyze the hydrolysis of the feedstock.
However, despite the foregoing advantages associated with high solids content slurries, their handling downstream of pretreatment can pose problems. For instance, in order for conventional stirred reactors to mix the highly viscous slurry effectively during enzymatic hydrolysis, a very large power input is required. Moreover, specialized high solids consistency pumps are needed to convey the high solids content slurry through the system. These requirements can significantly increase the capital and operating costs of the hydrolysis process.
There has been much effort devoted to hydrolysis reactor development, but it has focused primarily on dilute systems. WO 2006/063467 (Foody et al.) discloses the use of an unmixed upflow hydrolysis reactor for the enzymatic hydrolysis of a pretreated cellulosic feedstock with cellulase enzymes. The hydrolysis is conducted so that the upward velocity of the slurry is slow and the solid particles, which are denser than the bulk slurry, tend to flow upward more slowly than the liquor. The slow upward flow of the cellulose-containing solid particles retains the cellulose-containing solids and the bound cellulase enzymes in the reactor for a longer time than the liquid, thereby improving the conversion of the cellulose to glucose.
U.S. Pat. No. 5,258,293 (Lynd) discloses a method in which lignocellulosic feedstock and microorganisms are continuously introduced into a reaction vessel. Fluid is also continuously added from the bottom of the reaction vessel, but no mechanical agitation of the slurry occurs. As the reaction progresses, the lignocellulosic feedstock being digested tends to accumulate in a spatially non-homogenous layer, while the ethanol product rises to a top layer, where it is removed. The insoluble substrate accumulates in a bottom layer and can be withdrawn from the vessel. This arrangement results in a differential retention of the fermenting substrate, which allows for increased residence time in the reactor vessel.
In another approach, disclosed in U.S. Pat. No. 5,837,506 (Lynd), ethanol is produced using an intermittently agitated, perpetually fed bioreactor. Lignocellulosic slurry and microorganisms are added to a reactor; the mixture is then agitated, either by mechanical means or by fluid recirculation, for a specific time interval, after which it is allowed to settle. Ethanol is then removed from a top portion of the reactor, additional substrate is added, and the cycle continues.
In a similar method, Kleijntjens et al. (1986, Biotechnology Letters, 8:667-672) utilize an upflow reactor to ferment cellulose-containing substrate in the presence of C. thermocellum. The substrate slurry settles to form an aggregated fibre bed, which is accelerated by slow mechanical stirring. Substrate is added periodically, while liquid is continuously fed to the reactor. Ethanol product accumulates in a top layer, where it is removed from the reactor.
However, the methods described in U.S. Pat. Nos. 5,258,293 and 5,837,506 and Kleijntjens et al. (supra) may not be suitable for concentrated systems.
WO 2009/045651 (Hennessey et al.) discloses a process for saccharifying pretreated biomass at a high dry weight to produce fermentable sugars. The process of the invention uses a fed batch reactor system including multiple size reduction steps and thorough mixing in a standard vertical, agitated tank. Biomass is introduced into the vertical reactor tank equipped with an overhead agitator system such as a motor and shaft with one or more impellers.
US 2009/0098616 (Burke et al.) discloses a process in which a fine particulate stream of feedstock is subjected to a two-stage enzymatic hydrolysis process. The two-stage process allows for a reduction in the viscosity of the feedstock in the first stage and the production of a process stream that is rich in fermentable sugars in the second stage. Viscosity reduction is carried out in stirred reaction vessels and is thought to occur by the hydrolysis of xylan to soluble oligomers.
U.S. Pat. No. 5,733,758 (Nguyen) discloses an approach using a tower hydrolysis reactor comprising alternating mixed and unmixed zones. The solids loading of the slurry introduced to the hydrolysis is greater than 10 wt %. The slurry is moved upward in plug flow through the reactor and is intermittently mixed in the mixing zones, thus preventing channeling of liquid and ensuring uniform heat and mass transfer. As set forth therein, frequent and high initial mixing at the beginning of the hydrolysis when viscosity of the slurry is high is required to obtain as high a hydrolysis rate as is obtainable by continuous mixing.
U.S. Pat. No. 7,598,069 (Felby) discloses a method for hydrolyzing polysaccharide containing biomasses having a final dry matter content of above 20% involving mixing by a free fall type mixing that provides mechanical degradation of the biomass during hydrolysis. The mixer may be a drum mixer, a mixer with a rotary axis lifting the biomass or a similar mixing device utilizing a free fall principle. Such mixers are typically very large and significant power is required to rotate the vessel.
Despite these efforts, there is a need for more efficient and cost effective processes for enzymatically hydrolyzing cellulose in concentrated systems to obtain fermentable sugar. In particular, there is a need in the art to further reduce capital and operating costs associated with such processes so as to make them commercially viable.